Prochloron spp. are obligate cyanobacterial symbionts of many didemnid family ascidians. It has been proposed that the cyclic peptides of the patellamide class found in didemnid extracts are synthesized by Prochloron sp., but studies in which host and symbiont cells are separated and chemically analyzed to identify the biosynthetic source have yielded inconclusive results. As part of the Prochloron didemni sequencing project, patellamide biosynthetic genes were identified, and their function confirmed by heterologous expression of the whole pathway in Escherichia coli. The primary sequence of patellamides A and C is encoded on a single open reading frame that resembles a precursor peptide. This pre-patellamide is heterocyclized to form thiazole and oxazoline rings, and the peptide is cleaved to yield the two cyclic patellamides, A and C.
Marine invertebrates, particularly sponges and ascidians, are well known for their production of bioactive natural products (Newman et al. (2005) Mol. Cancer. Ther. 4, 333-342. A major hurdle in the development of many of these agents into drugs has been their supply, since collection or aquaculture of marine invertebrates pose many difficulties and may not be environmentally acceptable. Because marine invertebrate compounds often resemble molecules isolated from bacteria, many compounds are synthesized by symbiotic bacteria and not by the animals themselves (Faulkner et al. (1993) Gazz. Chim. Ital. 123, 301-307; Kobayashi et al. (1993) Chem. Rev. 93, 1753-1770; Sings et al. (1996) J. Ind. Microbiol. Biot. 17, 385-396; Haygood et al. (1999) J. Mol. Microbiol. Biot. 1, 33-34). Recently, these early speculations have been borne out in the cloning and sequencing of genes from two symbiotic natural product pathways (Piel et al. (2004) Proc. Natl. Acad. Sci. USA 101, 16222-16227; Hildebrand et al. (2004) Chem. Biol. 11, 1543-1552), opening a new era in marine natural products discovery and development.
Ascidians in the family Didemnidae contain numerous structural classes of cyclic peptides and harbor symbiotic cyanobacteria, Prochloron spp. (FIG. 15) (Withers et al. (1978) Phycologia 17, 167-171; Lewin, R. A. & Cheng, L. (1989) (Chapman and Hall, New York)). Despite nearly 30 years of attempts, Prochloron sp. have eluded cultivation and are thus considered to be obligate symbionts. Prochloron sp., unlike the vast majority of cyanobacteria but like plants, use both chlorophylls a and b for photosynthesis, lack phycobilins, and have plant-like thylakoids (Withers et al. (1978) Proc. Natl. Acad. Sci. USA 75, 2301-2305). The cells are relatively large for bacteria (10-20 □m in diameter). Prochloron has also been implicated in the biosynthesis of cyclic peptides isolated from whole didemnid ascidians. In early cell-separation studies, it was reported that the peptides were localized in Prochloron cells (Degnan et al. (1989) J. Med. Chem. 32, 1349-1354; Biard et al. (1990) J. Mar. Biol. Assoc. UK 70, 741-746), but a later investigation found the molecules distributed throughout the ascidian tunic, as well as in the cyanobacteria (Salomon, C. E. & Faulkner, D. J. (2002) J. Nat. Prod. 65, 689-692). Because of the unique biological and chemical features of the Prochloron-ascidian symbiosis, a project was initiated to sequence the genome of Prochloron didemni, isolated from the ascidian Lissoclinum patella. 
The patellamides and trunkamide (another didemnid product) are peptides that exemplify both the unique structural features and potent bioactivities of didemnid ascidian natural products (FIG. 15). Both groups have clinical usefulness, since patellamides are typically moderately cytotoxic, and patellamides B, C, and D reportedly reverse multidrug resistance (Williams et al. (1993) Cancer Lett. 71, 97-102; Fu et al (1998) J. Nat. Prod. 61, 1547-1551), while trunkamide was initially isolated because of specific and unusual activity against the multidrug resistant UO-31 renal cell line (Carroll, A. et al (1996) Aust. J. Chem. 49, 659-667). Patellamides are characteristically composed of pseudo-symmetrical, cyclic dimers, with each substructure having the sequence thiazole-nonpolar amino acid-oxazoline-nonpolar amino acid. Trunkamide and related molecules often contain proline, thiazolines, and prenylated serine and threonine derivatives. These features can result from either a ribosomal or a nonribosomal peptide biosynthetic pathway, since precedents exist for heterocyclization and cyclization in both cases (Gehring et al. (1998) Biochemistry 37, 11637-11650; et al. (2000) Nature 407, 215-218; Li et al. (1996) Science 274, 1188-1193; Solbiati et al. (1999) J. Bacteriol. 181, 2659-2662). The nonribosomal hypothesis of patellamide biosynthesis was investigated using a homology-based approach (Schmidt et al (2004) J. Nat. Prod. 67, 1341-1345). Only a single nonribosomal peptide synthetase (NRPS) gene was identified in fosmid clones, but the gene was found in only a few strains, and its presence did not correlate with patellamide production.
Bacterial secondary metabolites are bioactive small molecules that often find use as pharmaceuticals. (Newman et al. J. Nat. Prod. 66, 1022-1037 (2003)). Numerous studies of secondary metabolite biosynthetic genes have led to an increasing ability to synthesize new small molecules through rational pathway engineering (Floss J. Biotechnol. epub (2006); Walsh, C. T. ChemBioChem, 124-134 (2002)). Much of this capability comes from gene sequence comparison, in which the observation of evolution of these pathways has enabled engineering. Despite the advances, a weakness of this approach is that most described pathways are relatively distantly related, making an analysis of single evolutionary events difficult to discern. This difficulty is compounded by the large number of dedicated enzymatic steps (up to approximately 60 or so) commonly required to synthesize individual secondary metabolites.
Small, cyclic peptides are valuable pharmaceuticals, biotechnological products, and tools for scientific research (Davies, J. S. Amino Acids, Peptides and Proteins 2003, 34, 149-217). Cyclic peptides in general have advantages over their linear relatives in that they sample a more constricted conformational and configurational space. (Payne et al. Curr. Org. Chem. 2002, 6, 1221-1246). Stemming from this basic property, cyclic peptides often have stronger binding constants and favorable pharmacological properties such as resistance to proteases (Fairlie, D. P.; Tyndall, J. D. A.; Reid, R. C.; Wong, A. K.; Abbenante, G.; Scanlon, M. J.; March, D. R.; Bergman, D. A.; Chai, C. L. L.; Burkett, B. A. J. Med. Chem. 2000, 43, 1271-1281). Because of this, numerous investigators have developed means to produce arrays of small, cyclic peptides. Synthetic and enzymatic systems, as well as combinations of the two, have been used successfully on small and medium scale (Davies et al. J. Peptide Sci. 2003, 9, 471-501; Hahn et al. Proc. Nat. Acad. Sci. USA 2004, 101, 15585-15590). At the large scale, peptides in phage-display libraries have been cyclized via disulfide bonds or via semi-synthesis from the same libraries (Kehoe, J. W.; Kay, B. K. Chem. Rev. 2005, 105, 4056-4072; Ho, K. L.; Yusoff, K.; Seow, H. F.; Tan, W. S. J. Med. Virol. 2003, 69, 27-32).
There is a great need for new methods for making cyclic peptides, particularly for the manufacture of synthetic cyclic peptides for clinical investigations and therapeutic use, and for the production of cyclic peptide libraries that can be screened to identify cyclic peptides with a desired activity. What is needed in the art are methods for the in vivo construction of cyclic peptide libraries that are enzymatically cyclized at the C—N terminus.